Madsen K M, Zhang L, Abu Shamat A R, Siegfried S, Cha J H
Division of Nephrology, Hypertension and Transplantation, University of Florida College of Medicine, Gainesville, USA.
J Am Soc Nephrol. 1997 Jul;8(7):1043-53. doi: 10.1681/ASN.V871043.
Osteopontin is a secreted phosphoprotein that is expressed in the normal kidney and induced during various pathologic conditions associated with tubulointerstitial injury. However, the exact cellular location of osteopontin in the kidney has been a matter of controversy, and little is known about the role of osteopontin in the kidney. The purpose of this study was to establish the cellular and intracellular distribution of osteopontin in the rat kidney under normal conditions and after injection of a bacterial endotoxin lipopolysaccharide (LPS). Animals received injections of LPS or vehicle at different time intervals from 4 to 20 h before sacrifice. Kidneys were preserved by in vivo perfusion with paraformaldehyde-lysine-periodate (PLP) and processed for light and electron microscope immunocytochemistry using monoclonal antibodies to rat osteopontin. By light microscopy, immunostaining was observed in the descending thin limb and the papillary surface epithelium of both control and LPS-treated animals. After injection of LPS, osteopontin immunostaining was observed throughout the distal nephron and was also present in segments of the proximal tubule, where it was distributed in a punctate pattern. Staining was already present 4 h after injection of LPS and was maximal 6 h after injection. Electron microscopy revealed that osteopontin immunoreactivity in the descending thin limb and distal tubule cells was located in the Golgi apparatus and in small cytoplasmic vesicles, whereas in the proximal tubule labeling was observed in the vacuolar-lysosomal system. Western blot analysis demonstrated a band at approximately 70 kD and confirmed the increase in osteopontin expression after administration of LPS. These results demonstrate that osteopontin is constitutively expressed in cells of the descending thin limb and papillary surface epithelium and is induced throughout the distal tubule after administration of LPS.
骨桥蛋白是一种分泌型磷蛋白，在正常肾脏中表达，并在与肾小管间质损伤相关的各种病理状态下被诱导产生。然而，骨桥蛋白在肾脏中的具体细胞定位一直存在争议，人们对其在肾脏中的作用也知之甚少。本研究的目的是确定在正常条件下以及注射细菌内毒素脂多糖（LPS）后，大鼠肾脏中骨桥蛋白的细胞和细胞内分布情况。在处死动物前4至20小时的不同时间间隔，给动物注射LPS或赋形剂。通过用多聚甲醛 - 赖氨酸 - 高碘酸盐（PLP）进行体内灌注来保存肾脏，并使用抗大鼠骨桥蛋白的单克隆抗体进行光镜和电镜免疫细胞化学处理。光镜下，在对照动物和LPS处理动物的降支细段和乳头表面上皮中均观察到免疫染色。注射LPS后，在整个远端肾单位均观察到骨桥蛋白免疫染色，并且在近端小管节段中也存在，呈点状分布。注射LPS后4小时即出现染色，注射后6小时染色最强。电镜显示，降支细段和远端小管细胞中的骨桥蛋白免疫反应性位于高尔基体和小的细胞质囊泡中，而在近端小管中，标记物则出现在液泡 - 溶酶体系统中。蛋白质免疫印迹分析显示在约70kD处有一条带，并证实给予LPS后骨桥蛋白表达增加。这些结果表明，骨桥蛋白在降支细段和乳头表面上皮细胞中组成性表达，并在给予LPS后在整个远端小管中被诱导表达。
